An analysis of vertebrate remains from the middle Pleistocene open-air site of Holon was undertaken. The site is located in the coastal plain Quaternary Kurkar group near Tel Aviv. The faunal samples available to us were excavated in 1963, 1964 and 1970, and comprise 566 identifiable bones and teeth. Elephant (Palaoxodon antiquus), aurochs (Bos primigenius) and fallow deer (Dama mesopotamica) dominate the mammalian assemblage (93%) which also includes hippopotamus, wild boar, gazelle and tortoise. Studies of skeletal representation, bone surfaces and bone fragmentation suggest that while man broke the bones and extracted marrow from them, carnivores and rodents also visited the site (presumably after man’s departure) and chewed over a few of the bones. Finally both in prehistory and in a modern times chemico-physical weathering attacked most of them (fluvial dispersal). The results are discussed within the context of general issues concerning Acheulean subsistence strategy in the Near East.

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Rihet Pascal

Genetic susceptibility to P. falciparum in humans/ evolution of the human genome under selective pressure from malaria

The outcome of human malaria infection is thought to depend on socio-economic factors, on the type of transmission, and on parasite or host genetic factors.

Malaria is a complex disease and the analysis of various phenotypic variations related to infection should be helpful in dissecting the host genetic control of susceptibility. We have investigated genetics of blood infection levels and immune responses associated with resistance to malaria in an urban population living in Burkina Faso. We evidenced a protective role of cytophilic antibodies and a blocking role of noncytophilic antibodies. Furthermore, we detected familial aggregation of IgG subclass responses against various Plasmodium falciparum antigens and familial aggregation of blood infection levels. We further detect, in the same population, linkage between blood infection levels and chromosome 5q31-q33, which contains genes encoding cytokine involved in antibody production and in isotype switching towards some particular IgG subclasses. The identification of human genes controlling blood infection levels and protective immune responses may contribute to a better understanding of immune mechanisms and may provide new insights into the development of vaccination strategies.

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Dowling Thomas 1., Gilles André2, Smith Gerald .3

Does hybridization play a role in diversification of animals? A case study from catostomid fishes

While reticulate evolution has long been known to be important for plants, zoologists have generally considered introgressive hybridization to play little or no role in the evolution of animals. This latter view has recently been challenged; therefore, it is important to examine the potential role of hybridization in the evolution of animals. Fishes of the family Catostomidae (suckers) are well-suited for such tests because hybridization is common and several taxa have been hypothesized to have been generated by this process. We have examined the influence of hybridization by contrasting phylogenetic trees from different data sets. Thje lack of congruence between trees based on morphological characters and mitochondrial DNA (mtDNA) sequences is consistent with the impact of hybridization on this group. Conflicting lineages between trees often involved sympatric or geographically adjacent taxa that were often more similar for mtDNAs than expected from morphological characters. Sequences from the recombination activation gene (RAG) were consistent with the mtDNA tree but not the morphological tree, indicating that (1) both mtDNA and RAG genes provide evidence for historical introgression between lineages or (2) that morphological characteristics are not evolving as expected from the fossil record.

According to molecular phylogenetic data, in Lake Baikal a crown-like speciationoccured in several groups of molluscs and in several lineages of Limbriculidae (Oligochaeta).Interestingly, these events did not involve organisms like amphipods, fishes and Sergentia (Chironomidae, Diptera). Seemingly only species flocks of organisms boundto the bottom surface during whole life cycles underwent explosive speciation. No particular geologic events currently can be correlated with this evolutionary event. In order to elucidate the evolutionary mechanisms acting during that peculiar period respective molecular phylogenies were analyzed together with available data on macro-and microdistributions of recent species as well as some other their important ecological characteristics such as feeding preferences. Special procedure of data presentation have been developed for that purpose, when for example, distributiondata were presented in a tree-like form (« co-occurrance tree » in this case), which allowed to use maximum likelihood and topological statistic tests in order to choose between alternative hypotheses on the impact of the respective factors one volutionary process. In general it have been shown that para-patric mechanisms of speciation according to adaptation to narrow niches (micro-distribution patterns) played the major rôle in fast speciation. No evidence for such evolutionary mechanisms were obtained for more motile organisms dwelling at least temporally above the bottom surface.

European cyprinids have recently become the focus for molecular studies, namely addressing the evolutionary history of the family and trying to determine the validity of subfamilies. There is no consensus regarding the number and the monophyly of sufamilies, either from morphological (e. g. Arai, 1982; Chen et al., 1984; Howes, 1991) or molecular data ( Briolay et. al., 1998, ; Guilles et al., 1998; Zardoya and Doadrio, 1998, 1999). Howes (1991) recognise two main lineages of Cyprinidae: barbelled Barbini and barbel-lacking Leuciscinae, although it is unclear whether the presence of barbels is plesiomorphic or apomorphic. The first lineage is divided into three subfamilies (Cyprininae, Gobioninae and Rasborinae) and the latter in four subfamilies (Leuciscinae, Alburninae, Cultrinae and Acheilognatinae). More recently, phylogenetics studies based on the partial cytochrome b and 16sRNA sequences (Guilles et al., 1998) or complete cytocrome b gene sequences ( Briolay et. al., 1998, Zardoya and Doadrio, 1998, 1999) concerning mainly European cyprinids show somewhat discordance about the validity of these subfamilies: Zardoya and Doadrio (1999) considered them into two subfamilies – Cyprinidae (including barbins) and Leuciscinae (including cultrins, tincins, gobionins, phoxinins, and alburnins + leuciscins), while Guilles et al., (1998) showed that phoxinis are the sister group of alburninis + leuciscins. In these studies the relative position of Gobio and Tinca are unsolved or these taxa are considered Leuciscinae.

Briolay et al.(1998) considered the radiation of 6 subfamilies rather than a early split between barbelled and barbel-lacking lineages, meaning several appearances of barbels during cyprinid evolution. The present study is the first attempt to infer the evolutionary relationship of European, Asiatic and American Cyprinids based on a considerable number of representatives of the different genera in these different continents. This was done throughout the analysis of sequences of all cytochrome b and partial 16S RNA.

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Meyer Everett, Wheat Chris W. and Watt Ward B.

Levels of selection: toward explaining patterns of genetic variation at PGI among Colias butterfly species complexes

In studying the effects of morphological and genetic variation on Colias butterfly performance and fitness in the wild, we focus on the thermal phenotype (wing reflectance and fur thickness) and biochemical performance of the glycolytic enzyme phosphoglucose isomerase (PGI). Here we present new data on newly surveyed species, C. meadii and reviving older data for a comparative analysis of the biogeography of their genetic variation. The North American lowland complex, comprised of Colias eurytheme and C. p. eriphyle, is multivoltine and found throughout the continent. C. meadii is univoltine and inhabits subalpine/alpine meadows throughout the Rocky Mountains. Using morphological measurements of Colias thermal phenotype and three decades of phosphoglucose isomerease (PGI) allozyme data, we present data documenting significant differences in biogeographic genetic variation between these two species. Lowland populations maintain PGI allele frequency homogeneity over large geographic distances (1200 km), with thermal phenotypic modulation among seasonal generations. Above and below timberline C. meadii populations show significant differences in both PGI allele frequencies and thermal phenotype over narrow geographic distances (1-10 km), in both continuous clinal and isolated populations. Unifying this data with previous work on the biochemical, field performance, and fitness consequences of this variation further documents the incredible thermal sensitivity of Colias. Comparisons between these two species complexes provide insight into the interaction between differing levels of the genetic architecture of Colias, allowing us to ask how metabolic performance interacts with the thermal phenotype in different environments.

Transposable elements are short but complex pieces of DNA or RNA containing a streamlined minimal-genome with the capacity for its selfish replication in a foreign genomic environment. Cis-regulatory sections within the elements orchestrate tempo and mode of TE expression. Proteins encoded by TEs mainly direct their own propagation within the genome by recruitment of host-encoded factors. On the other hand TE-encoded proteins harbor a very attractive repertoire of functional abilities for a cell. These proteins mediate excision, replication and integration of defined DNA fragments. Furthermore, some of these proteins are able to manipulate important host factors by altering their original function.

Thus, if the host genome succeeds in domesticating such TE-encoded proteins by taming their « anarchistic behavior » such an event can be considered as an important evolutionary innovation for his own benefit.

In fact, the domestication of TE-derived cis-regulatory modules and protein coding sections took place repeatedly in the course of genome evolution. We will present prominent cases that impressively demonstrate the beneficial impact of TEs on host biology over evolutionary time. Furthermore, we will propose that molecular domestication might be considered as a resumption of the same evolutionary process that drove the transition from « primitive genomes » to « modern » ones at the early dawn of life: the adaptive integration of a short piece of autonomous DNA into a complex regulatory network.

RT-PCR analysis of mRNAs from the lupus-prone NZW mouse indicated that they differ in the lengths of their 5′ untranslated regions (UTR). By sequence analysis, we determined that this size difference is the result of the presence in the 2nd intron of a cluster of short interspersed nucleotide elements (SINEs), in which a B1 element provides cryptic splice sites. The functional consequences of aberrantly spliced mRNAs are supported by a lower upregulation of CD22 membrane expression on activated B cells from strains bearing the Cd22a allele. Studies aimed at determining the form of this polymorphism in the genus Mus revealed that it was distributed across three species M. spretus, M. macedonicus and M. spicilegus, and five different M. musculus subspecies, but with a low frequency in M. m. domesticus, suggesting that an actual deletion event might have occurred within the nascent M. m. domesticus subspecies. Polymorphism described here is evidence for the potential of the nonviral B1 family of retroposons to affect the structure and expression of genes.

Despite the accumulation of sequence information sampling from a broad spectrum of phyla, newly sequenced genomes continue to reveal a high proportion (50% to 30%) of uncharacterized genes, including a significant number of strictly « orphan » genes, i.e. putative ORFs without any resemblance to previously determined protein-coding sequences. Most genes found in databases have only been predicted by computer methods and have never been experimentally validated. While theoretical evolutionary arguments support the reality of genes when homologues are found in a variety of distant species, this is not the case for orphan genes. Here, we report the direct RT-PCR assay of 25 strictly orphan ORFs of Escherichia coli .

Two growth conditions, exponential and stationary phases, were tested. Transcripts were identified for a total of 19 orphan genes, with 2 genes found to be expressed in only one of the two growth conditions. Our results suggest that a vast majority of E. coli ORFs presently annotated as « hypothetical » correspond to bona fide genes. By extension this implies that randomly occurring « junk » ORFs have been actively counter selected during the evolution of the dense E. coli genome. Expression, protein purification and 3-D structure determination by X-ray diffraction is currently attempted for 5 E.coli ORFans. We will report on the progress of this exploratory « structural genomics ».

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Joseph P. A., Fichant G., Quentin Y. and Denizot F.

Conservation of a genetic link between two-component regulatory systems and ABC permeases in a particular group of Bacteria.

LCB – IBSM – CNRS, 31 chemin Joseph Aiguier, 13009 Marseille

When comparing the location of two-component systems and ABC transport systems on the Bacillus subtilis chromosome, nine cases of close proximities between genes encoding these two systems were found. In three instances, i) the organization of the different genes is conserved, ii) each of the functionally related components showed high similarities not only at the protein level but also at the nucleic level. These suggest that the three genetically linked systems were obtained by successive duplications of the entire chromosomal region. In order to detect if such a genetic association is found in other bacteria, we looked for the presence and proximity of similar systems in other bacterial genomes.

We used each components (Nucleotide Binding Domains (NBD), Membrane Spanning Domain (MSD), Histidine Kinase (HK) and Response Regulator (RR)) of the three associated systems, as query in blast program to scan genomes either entirely sequenced (blast P2) or from which sequence was in progress (tblastn). When a protein presenting similarities was detected, the vicinity of its corresponding gene was analyzed for the presence of genes encoding the other components. Finally, all detected proteins belonging to the same functional class were aligned using ClustalW and trees were computed using the Neighbor Joining method. The two component and ABC permeases similar to the duplicated systems detected in B. subtilis were only found in other low G+C Gram positive bacteria as Clostridium acetobutilicum, Clostridium difficile, Staphylococcusaureus, Streptococcus mutant and Streptococcuspneumoniae but not in the mycoplasma. Evolutionary tree reveals that all the RR found constitute a sub-family of the OmpR response regulator family. In addition, analysis of the gene organization shows that each of them is genetically linked to an ABC permease. The NBD of these ABC transport systems constitute a particular class in the NBD sub-family 9 (http://ir2lcb.cnrs-mrs.fr/ABCdb/). The association between two-component and ABC transport systems might have a common origin in the Bacillus/Clostridium group. All these observations suggest that both systems might undergo functionally constraints and be involved in the same physiological process.

Leuciscus alburnoides is an endemic cyprinid fish found in Iberian inland waters, which comprises diploid and polyploid forms and is involved in mechanisms of unisexual reproduction. Recent studies on L. alburnoides revealed the existence of several features, e.g. recombination of homospecific genomes in triploid females, ploidy shifting, participation of hybrid males in perpetuation of the complex and meiosis in tetraploid hybrid males.

These features make it unusual in the context of unisexual vertebrates, and a promising system for understanding how lineages may compensate for the disadvantages of unisexuality. The present study has the objective of enhancing knowledge about the genetic structure, dynamics and evolutionary potential of the L. alburnoides complex by: 1) finding genetic markers for genomic characterisation of the distinct forms and investigation of the sex-determination system; 2) further examining the reproductive modes of L. alburnoides.

To assess the first question, a genetic survey of unisexual populations from the Tejo and Guadiana basins have been performed using microsatellite markers and chromosome banding. The implementation of the second objective involve experimental crosses, and the analysis of progenitors and respective progenies using the above mentioned genetic markers. The study of such fish complex is an excellent opportunity to provide some insight into the evolutionary significance of unisexuality and of the role of hybridization and polyploidy in animal evolution.

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Thomas Stach

Modern morphological investigations on the « phylotypic stage » of the European amphioxus (Branchiostoma lanceolatum)

The early larval stage of the European lancelet Branchiostoma lanceolatum has been investigated by means of immunohistochemistry and transmission electronmicroscopy. Monoclonal antibody labelling against acetylated tubulin in combination with confocal laser scanning microscopy revealed the detailed three-dimensional arrangement of nervetracts and ciliary structures. The preoral pit, a homologous structure to craniate Rathke’s pouch, is shown to be innervated.

A complex nerve-plexus underlying a rostral epidermal papilla is discribed for the first time.No segmental structures were revealed in the nervous system by this method. The well known asymmetry of the larval stage in amphioxus is also seen in the peripheral nerves. Homologies to craniate and tunicate structures and possible functions are considered. The concept of the « phylotypic stage » is briefly discussed.

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Thomas Stach

Development of trunk muscles and associated structures in Branchiostoma lanceolatum

Developmental stages of Branchiostoma lanceolatum were examined, combining light and electron microscopical techniques. Serial sections of specimens were prepared, and the ontogeny of the mesodermal segments studied from the neurula to the early larval stage. The origin of the mesoderm from the archenteron was confirmed. The close contact between the neural tube cells and the prospective myocytes is established early in neurula stages, before the myocytes are differentiated. Two cell types are present in a mesodermal segment from neurula stages on: the medial myoblasts, which develop into the myocytes (= myofibrillae = myolamellae), and a lateral cell group, which becomes coelothelial during development. The latter cell group is possibly homologous to the craniate dermatome. Tissue resembling the sclerotome of craniates was not observed. A low rate of mitosis suggests, that differentiation rather then cell division and cellular migration, is the preferred developmental mode in the mesoderm of cephalochordates in this stages. In contrast to adult individuals only one type of myocytes is present in early larvae.

They are similar to the deep lamellae of the adults. It is suggested tentatively, that the lack in superficial lamellae is correlated functionally to the preference of the larvae to « hover » by ciliar propulsion, whereas adults rely on muscular propulsion entirely. In addition the number of myocytes per segment is considerably lower in developmental stages than in the adults: 15 – 25 versus >1000.

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Laudet Vincent and escriva Hector

Nuclear receptors in amphioxus: implications in the evolution of new morphological structures

Nuclear receptors are a superfamily of ligand activated transcription factors that are conserved through evolution of all metazoans. Our phylogenetical studies suggest that two waves of gene duplications took place during the evolution of the superfamily. First, giving a subdivision in six subfamilies and second (specifically in vertebrates) giving several paralogues for each gene.

We have focussed our attention in the role of such duplications in the apparition of new structures and metabolical pathways in vertebrates. Thus, our system model is amphioxus which is placed before the specific gene duplication of vertebrates but present a pattern of development comparable to them, although somewhat simpler. We have choosen two nuclear receptors in our sudy (the retinoic acid receptor, RAR, and the orphan receptor TR2/4), due to their implication in the development of different structures in vertebrates. We have foccused the study in the development of the gill slits as the evolutive precursors of an important morphological structure, the jaws.

We present here, expression, and functional studies of RAR, and TR2/4 that demonstrate their role in the formation of the amphioxus gill slits.

Department of Genetic Information, Division of Molecular Life Science, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.

Most of the genes located in the Major Histocompatibility Complex (MHC) region result from cis duplication and exon shuffling, and constitute several multigenic families. The exon shuffling occurred close to and before the radiation of jawed vertebrates. This conclusion is based on the fact that the genes issued from such rearrangements (in particular MHC class I and class II genes) and their corresponding function are only found in jawed vertebrates.

In the vicinity of these genes forming multigenic families are found other genes called anchor genes, which are single copy in this portion of genome. It should be noted that the anchor genes are stable in evolution, and have evolved more slowly than genes forming multigenic families. Indeed unlike genes forming multigenic families, anchor genes are found in non-vertebrate species. Some of the MHC anchor genes are found in three other genomic regions (representing paralogous regions). These could be the remains of two large scale duplications that occurred after the separation of cephalochordate ancestors and those of jawed vertebrates. Our hypothesis is that the genetic information contained in these regions became redundant in those species whose ancestor genomes underwent two rounds of duplication.

Genomic rearrangement (duplication, exon shuffling, etc.) was permitted in this region, even if important genes may have been deleted during this process, with no deleterious effect. These different rounds of cis-duplication and exon shuffling allowed the emergence of new genes participating in novel biological functions i.e. adaptive immune responses. As a corollary, such chromosomal rearrangements had to be absent in species where large scale duplications did not occur, since gene deletions would have been deleterious.

This hypothesis is currently tested in the laboratory by analysis of the amphioxus MHC-like region. As this region was probably not quadruplicated in this species lineage, the formation of the MHC multigenic families have probably not been permitted. So it may be possible to find genes derived from ancestral genes that gave rise to the multigenic families of the MHC, in synteny with MHC anchor genes.

Several anchor genes have already been cloned in the amphioxus, as well as the corresponding genomic regions. Phylogenetic analysis of these genes indicate that the amphioxus cloned anchor genes are orthologous to all the vertebrate paralogs. This supports the occurrence of large scale duplications after the separation of cephalochordates and vertebrates. Furthermore, analysis of the genomic regions around some of these genes shows that, at least, several vertebrate syntenies are preserved in the amphioxus.

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Cañestro Cristian, Albalat Ricard and Gonzàlez-Duarte Roser.

Insights of a gene family expansion through the vertebrate evolution: the ADH3 of amphioxus.

The increase in the number of genes during animal evolution illustrates gene family expansions and the acquisition of new phenotypic traits at key evolutionary stages. Genome duplications, which occurred at the base of the vertebrate radiation appear to be linked to the extensive multiplicity of the protein families already described in vertebrates. As a gene family model we have focused in the alcohol dehydrogenase (ADH), which belongs to a complex enzyme system: the medium-chain dehydrogenases/reductases (MDR). Different classes of ADH can be found in the vertebrate lineage, of which at least six are presently known in mammals. Class 1, the classical ethanol-active form, and class 3, the glutathione-dependent formaldehyde dehydrogenase, are the ones furthest investigated in different animal clades.

We have analized the amphioxus (B. floridae and B. lanceolatum) ADH3 genomic structure as well as the full cDNA sequence. Southern blot analysis indicates that amphioxus ADH3 gene is encoded by a single gene located in the methylated fraction of the genome. No ethanol-active ADH classes have been detected. A high level of intra-specific variability has been observed in this genomic region, due to imperfect tandem repeats expanding the intron-exon boundaries. Moreover, further experiments suggested intra-individual somatic polymorphism concerning repeat number.

Northern blot analysis revealed a single 1.4 kb transcript. In situ hybridizations showed Adh3 expression restricted to particular embryonic cell types, in contrast with the ubiquitous expression already reported in mouse and the proposed housekeeping function for this gene. Evolutionary studies date the ADH3-ADH1 duplication 500 million years ago, more linked to the « isoform burst » before the gnathostome radiation than with the two rounds of genome duplication postulated at the base of vertebrate evolution.

The ADH3 protein could also be used as a molecular clock to date the « Cambrian explosion » in the late Neoproterozoic. Under the ADH3-clock protostomes-deuterostomes and cephalochordates-vertebrates diverged around 740 and 690 million years ago, respectively.

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Pontarotti Pierre

Did large scale duplication occurred in our chordate ancestors ?

Phylogénomique. INSERM Unité 119, 27 bd Leï Roure, 13009 Marseille

We tested the hypothesis that different gene ancestors duplicated at the same time around 600 million years ago before the jaw vertebrate radiation and most likely this occurred en block . We focused on anchor genes present in the MHC and its three paralogous region ( see Laurent Abi Rached abstract) For that, multiple alignments were constructed using Clustal X on selected gene families. To determine the major groups of paralogous/orthologous genes of the different sequences obtained from Genbank, phylogenetic trees were constructed based on a Poisson Correction distance with a pairwise deletion comparison. All the trees are rooted on midpoint. After that, the groups were distinguished by high distance values and supported by high Bootstrap Proportion .These groups were then analyzed separately using Gu’s program. (see André Gilles Abstract). We found a divergence time of around 600 M years ago by this method. Orthologs of these genes have been cloned in amphioxus (see Laurent Abi Rached abstract) and phylogenetic analysis is congruent.

These results are consistent with large scale genome duplication of the MHC-like region prior to the divergence of the jawless and jawed vertebrates; however we cannot draw this conclusion at the whole genome level, and the main message of this presentation will be that this kind of careful and laborious analysis should be undertaken on different genome portions to decide whether or not whole genome duplications occurred.

Barnacles (Cirripedes) are crustaceans highly modified by a fixed way of life. Contrary to most crustaceans, they never develop a complete abdomen. Together with other morphological characters, this weird feature constitutes a shared characteristic (synapomorphy) of the whole Cirripedia. As an attempt to understand the genetic basis of this peculiar character, we undertook a study of genes involved in the body design in other arthropods.

Two engrailed genes are present in cirripedes. Their expression pattern shows two cephalic stripes corresponding to the maxillulary and maxillary segments, and six stripes corresponding to the six thoracic segments. Unexpectedly, five more small stripes are located between the sixth thoracic segment and the telson. We interpret this pattern as a clue for a vestigial abdomen. This vestigial abdomen can be seen even at early larval stages as a group of small cells separated from the large aligned thoracic cells. The vestigial abdomen is moved dorsally by the growth of the thorax.

In both protostomes and deuterostomes the caudal gene is expressed at the posterior end of the body. It has even been proposed that it could be the homeotic gene corresponding to this part of the anterior to posterior axis. We isolated a partial cDNA sequence of the caudal gene of the rhizocephalan cirripede Sacculina carcini, and examined the expression of this gene by in situ hybridization during embryonic and larval development. The Sacculina caudal gene is expressed early in embryogenesis, in a region that eventually gives rise to the caudal papilla. During larval development, caudal expression is quite dynamic. It is first expressed in the whole thorax, and eventually restricts itself to the posterior end of the thorax. Noteworthy, caudal is never expressed in the vestigial abdomen.

We examined the Hox repertoire of three species representative of the three cirripede orders. We failed to reveal the presence of an abdominalA orthologue, whereas it was found in an ascothoracid species. Ascothoracids, assumed to be the sister-group of Cirripedia, possess a six-segmented thorax like cirripedes, followed by an abdomen composed of four to five segments.

We conclude that a part of the developmental genetic program needed to form an abdomen is still present in cirripedes (engrailed), but that another part might have been lost (abdominalA) or impaired (caudal) during evolution. The lack of expression of caudal in the cirripede vestigial abdomen may be compared to the effects of a dominant negative form of the gene in ascidian embryos, resulting in defective tail formation. This suggest a conserved morphogenetic function of caudal in both protostomes and deuterostomes in posterior elongation.

The Drosophila homeotic gene abdominal-A is important for developpemental decisions in the anterior abdomen. Insects vary considerably with respect to abdominal morphology, and changes in the function of homeotic selector gene(s) and / or downstream genes under their control presumably have been important for the evolution of these differences.

The phenotype of the Myrmicinae ants is sugestive of a natural homeotic transformation like same mutations in abdominal-A of Drosophila or in abdominal-A ortholog of Tribolium. Here we present the organisation of abdominal-A ortholog of Myrmica rubra and the sequences of its predicted proteins.

T-box genes have begun recently to receive much attention as a new family of transcription factor with important roles in metazoan development. These factors are characterized by the sharing of a highly conserved DNA-binding domain of about 200 aa, the T-domain (encoded by the T-box). The most famous member of the T-box family, Brachyury (the T gene), has a critical role in mesoderm specification and the gastrulation process in vertebrates. Among triploblasts, homologues are known from urochordates, hemichordates, echinoderms, and Drosophila. Expression occurs in various cell and tissue types but seems to be correlated with the existence of invaginating structures. Recently, two Brachyury homologues have been isolated from the cnidarian Hydra vulgaris. One of them, HyBra1, is expressed in the endoderm of the hypostome (oral region) and appears to play a role in head formation. We have cloned and characterized a brachyury homologue from the calcareous sponge Syconraphanus. This is the first member of the T-box genes family reported from sponges, the most basal metazoans. Evolutionary implications of data obtained on this gene will be discussed.

The LMP2, LMP7 and MECL-1 genes code for b-type subunits of the proteasome, a multimeric complex that degrades proteins into peptides as part of the major histocompatibility complex (MHC) class I-mediated antigen presenting pathway. These gene products are up-regulated in response to infection by interferon g and replace the corresponding constitutively expressed subunits (X, Y and Z) during the immune response. In mammals, the LMP2 and LMP7 genes both reside within the class II region of the MHC (6p21.3), whilst MECL-1 is located at 16q22.1.

This linkage relationship is maintained in other vertebrates, although so far there is no data as to the relative location of MECL-1. We have identified all three interferon g regulated b-type proteasome subunits in Fugu, which are present as a cluster within the Fugu MHC Class I region. In this species, the proteasome subunit organisation represents a more primitive arrangement, supporting the theory that these genes arose from a tandem triplication event from a single primordial X/Y/Z-like gene to produce X, Y and Z, followed by whole chromosome duplication event to produce two separate loci containing X,Y,Z and LMP2, LMP7 and MECL-1. Also within this cluster is an LMP2-like subunit (which seems specific to all teleosts tested so far) and a closely linked LMP7 pseudogene, indicating that within Fugu and potentially other teleosts, there has been an additional regional duplication involving these genes. A brief overview will also be given of the MHC region in Fugu.

The coding fraction of human genes is estimated to represent less than 5% of the genome and is difficult to identify accurately and exhaustively. However, it is well known that sequence conservation in genes between distant species can be used to identify such coding regions. Here we report on a method to identify human genes by virtue of their conservation in the Tetraodon nigroviridis genome, a pufferfish distant by approximately 400 million years from humans. The basis for this work is the sequence of 33 % of the Tetraodon genome in single reads of average length ~900 bp. We show that a corresponding fraction (30%) of human exons can be rapidly and specifically detected in anonymous DNA with less than 1% false predictions. Calibration was performed on annotated pufferfish and human genes, to define the conditions in which the BLAST algorithm will rapidly generate alignments with a high specificity and sensitivity in coding regions. A classification based on length and percentage of identity was subsequently performed to distinguish alignments that overlap exons from those that overlap introns. Results show that alignments that overlap exons respect a precise definition that can now be applied to any human DNA of unknown gene content to predict the position of coding conserved regions. This tool, called Exofish (Exon Finding by Sequence Homology) has been applied to the entire sequence of human chromosome 22, to the december 1999 version of the human working draft sequence, and to Unigene. Results indicate that the human genome contains between 28,000 and 34,000 genes. In addition, Exofish shows that sequences representing Unigene clusters contains less than 40% of the coding fraction of the human genome.

Exofish is therefore a tool that allows – for the first time – comparisons between very large fractions of vertebrate genomes in all 6 frames. It represents a powerful tool to identify genes in human genomic DNA and to estimate the coding fraction in cDNA collections. Exofish is available at http://www.genoscope.cns.fr/exofish

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Laloui Wassila

Analysis of genes coding for small subunit rRNA sequences of Phaeoacremonium species isolated from Vitis vinifera.

A molecular analysis of Phaeoacremonium species isolated grapvine was undertaken from sequences of the small subunit rRNA, and the species placed within the overall Ascomycete classification. Neighbour-Joining analysis showed that the genus Phaeoacremonium is polypheletic. Great divergence is showed between Phaeoacremoniumchlamydosporum, Phaeoacremonuim aleophilum and Phaeoacremonium parasiticum. P. chlamydosporum appears to the closely related to the Chaetothyriales and the species P. parasiticum, and P. aleophilum were placed close to the Pyrenomycetes.

Identification of VEGFR (Vascular Endothelial Growth Factor Receptor) related genes in the Ecdysozoan phylum and their characterization in C. elegans.

INSERM Unité 119, 27 bd Leï Roure, 13009 Marseille, France

We made a systematic analysis of the tyrosine kinase receptors (RTKs) in three metazoan genomes which have almost been completely sequenced, with a special emphasis on the VEGFR family.

In humans, 3 VEGFR have been described. Because RTKs have been particularly well studied, no other member will be detected in the part of the genome which is not yet sequenced. Last year, we presented the description of a family of 4 genes encoding related receptors in Caenorhabditis. elegans (T17A3.1, T17A3.8, F59F3.1, F59F3.5) and concluded to a specific expansion of this gene family in the nematode. This hypothesis was recently reinforced by the analysis of the Drosophila melanogaster genome which revealed the presence of only one putative VEGFR (CG8222), suggesting the existence of a single common ancestor of all VEGFR genes in the Ecdysozoan phylum.

In mammals, VEGFRs play a key role in vasculature formation thereby making intriguing their presence in nematodes. No RTK related to mammalian class III RTKs (PDGFR, FLT3…), close to VEGFRs were found in C. elegans and Drosophila. This class of RTK may have derived from VEGFRs in the vertebrate phylum and may represent a case of exaptation.

Comparative analysis of genomes and functions indicates that molecular cascades are often conserved between species. We will use C. elegans to study the cascade of events which occurred after the interaction of VEGFs with their receptors using biochemical and genetic tools.